Diiron magnesium tetraoxide

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics, other

Remarks:

Bioaccessibility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-26 to 2015-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media

Deviations:

yes

Remarks:

Bioaccessibility testing: loading of 100 mg/L; five artificial physiological media agitated at 100 rpm, at 37 °C ± 2 °C; sampling after 2 h and 24 h; determination of Fe and Mg concentrations after filtration by ICP-OES.

Principles of method if other than guideline:

The test was performed on the basis of OECD Series on Testing and Assessment No. 29 as well as according to the bioaccessibility test protocol provided by the monitor. The bioaccessibility protocol has been developed on the basis of relevant published methods ([1], [2], [3], [4] and [5]).

The aim of this test was to assess the dissolution of the pigment IIPC-2018-008 (Magnesium ferrite) in the artificial physiological media GST, GMB, ALF, ASW and PBS. The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. a substance entering the human body by ingestion or by inhalation.

Five different artificial physiological media with a single loading of test substance of 100 mg/Lwere used. The measurement of dissolved iron and magnesium concentrations after filtration were performed by ICP-OES. Samples were taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C. The study was performed in triplicate with two additional method blanks per medium.

[1] Hanawa T. 2004. Metal ion release from metal implants. Materials Science and Engineering C 24: 745-752.
[2] Stopford W., Turner J., Cappelini D., Brock T. 2004. Bioaccessibility testing of cobalt compounds. Journal of Environmental Monitoring 5: 675-680.
[3] Midander K., et al. 2007. In vitro studies of copper release from powder particles in synthetic biological media. Environmental Pollution 145: 51-59.
[4] European standard 1998. Test method for release of nickel from products intended to come into direct and prolonged contact with the skin (EN 1811)
[5] ASTM 2003. Standard test method for determining extractability of metals from art materials. ASTM D5517-03.

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro (simulated human body fluids)

Details on test animals or test system and environmental conditions:

Test principle in brief:
- five different artificial physiological media,
- single loading of test substance of 100 mg/L,
- samples taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C,
- two method blanks per artificial media were tested; measurement (by ICP-OES) of dissolved iron and magnesium after filtration
- the study was performed in triplicate

The aim of this test was to assess the dissolution of IPC-2018-008 (Magnesium ferrite) in five artificial physiological media: Artificial lysosomal fluid (ALF, pH = 4.5), Artificial sweat solution (ASW, pH = 6.5), Gamble´s solution (GMB, pH = 7.4), Artificial gastric fluid (GST, pH = 1.5), Phosphate buffered saline (PBS, pH = 7.4). The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. a substance entering the human body by ingestion into the gastrointestinal tract and by inhalation.

Duration and frequency of treatment / exposure:

Samples were taken after 2 h and 24 h.

Dose / conc.:

100 other: mg of the test item /L artificial media

Details on study design:

Reagents
The water (resistivity >18 MΩ·cm.) used for this test was purified with a Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany.
- Nitric acid - “Supra” quality (ROTIPURAN® supplied by Roth, Karlsruhe, Germany).
- Hydrochloric acid – “instra-analyzed plus” quality (J.T. Baker, Griesheim, Germany).
- Sodiumhydroxide – pro Analysis quality (Chemsolute, Th. Geyer, Renningen, Germany)

Metal analysis
- Standards:
- Certified reference materials: As quality control standards TM-25.4 (lot no. 0914) and TMDA-53.3 (lot no. 0914) obtained from Environment Canada and a multielement standard (Merck XXI, lot no. HC42984673 and HC55190098, Darmstadt, Germany) were analysed for total dissolved iron and magnesium by ICP-OES along with the samples to determine the accuracy of the applied analytical method. Furthermore the calibration solutions were measured along with the ICP-OES measurements as recalibration standards.

Instrumental and analytical set-up for the ICP-OES instrument:
Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer from Agilent
Spray chamber: Glass cyclonic spray chamber from Agilent
Carrier gas flow: 0.75 L/min
RF power: 1200W
Wavelengths:
Fe: 234.350 nm, 238.204 nm, 239.563 nm, 240.489 nm, 241.052 nm, 258.588 nm, 259.837 nm, 259.940 nm, 261.157 nm, 263.105 nm and 275.574 nm
Mg: 202.548 nm, 206.200 nm and 213.857 nm

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645):
LOD: 3 x standard deviation of calibration blank/slope of the calibration
LOQ: 3 x LOD

Calibration: blank, 1 μg/L, 2.5 μg/l, 5 μg/L, 7.5 μg/L, 10 μg/L, 25 μg/L, 50 μg/L, 75 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 750 μg/L and 1000 μg/L.
Correlation coefficients (r): at least 0.999631

Details on dosing and sampling:

Loading:
Detailed loadings of the test vessels are given in "Any other information on materials and methods incl. tables".

Toxicokinetic parameters:

other: bioaccessibility

Remarks:

Dissolution of Fe at a loading of 0.1 g/L: ALF (pH 4.5) 24h: 428 ± 2.25 µg/L. Dissolution of Mg at a loading of 0.1 g/L: ALF (pH 4.5) 24h: 4.46 ± 3.36 µg/L

Bioaccessibility (or Bioavailability) testing results:

Concentration of dissolved iron in artificial physiological media.
Total Fe ± SD in sample vessels without method blank subtraction
- GST 2h:74.6 ± 8.73 µg/L (all method blanks below LOD/LOQ)
- GST 24h: 468 ± 18.6 µg/L (all method blanks below LOD/LOQ)
- GMB 2h: 0.264 ± 0.212 only one sample above LOQ, five samples below LOD (all method blanks below LOD)
- GMB 24h: All samples below LOD (all method blanks below LOD)
- ALF 2h: 66.9 ± 4.50 µg/L (all method blanks below LOD)
- ALF 24h:105 ± 2.46 µg/L (all method blanks below LOD)
- ASW 2h: All samples below LOD (all method blanks below LOD)
- ASW 24h: 0.795 ± 1.11 only one sample above LOQ, five samples below LOD (all method blanks below LOD)
- PBS 2h: All samples below LOD (all method blanks below LOD)
- PBS 24h: All samples below LOD (all method blanks below LOD)

Concentration of dissolved zinc in artificial physiological media.
Total Zn ± SD in sample vessels with mean method blank subtraction (mean of 2 h and/or 24 h)
- GST 2h: 3.26 ± 0.63 µg/L (no subtraction-> negative value, method blanks: 4.26 ± 2.95 µg/L)
- GST 24h: 2.17 ± 0.51 µg/L (method blanks: 5.47 ± 3.57 µg/L)
- GMB 2h: All samples below LOD (all method blanks below LOD)
- GMB 24h: All samples below LOD/LOQ (all method blanks below LOD)
- ALF 2h: 2.18 ± 1.51 µg/L (method blanks: 2.51 ± 0.26 µg/L)
- ALF 24h: 1.07 ± 0.41 µg/L (method blanks: 3.02 ± 1.12 µg/L)
- ASW 2h: 0.55 ± 0.23 µg/L (no subtraction-> negative value; method blanks: 1.47 ± 0.52 µg/L)
- ASW 24h:0.442 ± 0.33 µg/L (no subtraction-> negative value, method blanks: 2.20 ± 0.58 µg/L)
- PBS 2h: All samples below LOD (all method blanks below LOD)
- PBS 24h: All samples below LOD/LOQ (all method blanks below LOD)


Concentration of dissolved magnesium in artificial physiological media.
Total Mg ± SD in sample vessels with mean method blank subtraction (mean of 2 h and/or 24 h)

-GST 2h: 174 ± 3.09 μg/L
-GST 24h: 242 ± 3.96 μg/L
-GMB 2h: 153 ± 48.8 μg/L
-GMB 24h: 138 ± 18.4 μg/L
-ALF 2h: 297 ± 105 μg/L
-ALF 24h: 23.2 μg/L
-ASW 2h: 78.0 ± 5.75 μg/L
-ASW 24h: 167 ± 6.31 μg/L
-PBS 2h: 110 ± 2.94 μg/L
-PBS 24h: 161 ± 2.05 μg/L

Method validation summary ICP-OES

validation parameter	Results	Comment
Selectivity	similar data with different wavelengths for ICP-OES method	-
Linearity	applied calibration functions were linear	coefficient at least 0.999631
Limit of detection	Fe: 0.201 – 1.36 µg/L Mg: 0.054 – 0.491 µg/L
Limit of quantification	Fe: 0.603 – 4.07 µg/L Mg: 0.163 – 1.47 µg/L
Method blanks	Fe: - GST: All method blanks below LOD/LOQ - GMB, ASW, ALF, PBS: All method blanks below LOD Mg: - GST 2 h: 4.26 ± 2.95 µg/L; 24 h: 5.47 ± 3.57 µg/L - GMB, PBS: all method blanks below LOD - ALF 2 h: 2.51 ± 0.26 µg/L; 24 h: 3.02 ± 1.12 µg/L - ASW 2 h: 1.47 ± 0.52 µg/L; 24 h 2.20 ± 0.58 µg/L	-
Accuracy measurement / Reproducibility testsample and mass balance measurements	Mean recovery for CRM TM-25.4 (dilution factor 10): Fe: 104 ± 13 (n = 26) Mg: 105 ± 10 (n = 23)	Low concentration range (certified with 31.0 µg Fe/L, diluted 3.10 µg Fe/L; 44.5 µg Mg/L, diluted 4.45 µg Mg/L)
Accuracy measurement / Reproducibility testsample and mass balance measurements	Mean recovery for CRM TMDA-53.3 (dilution factor 10): Fe: 101 ± 3.4 (n = 30) Mg: 100 ± 5.1 (n = 30)	Low to mid concentration range (certified with 325 µg Fe/L, diluted 32.5 µg Fe/L; 386 µg Mg/L, diluted 38.6 µg Mg/L)
Truenesstestsample measurements	Quality control standard (Merck XXI): Fe: 105 ± 0.7 (n = 3) Mg: 105 ± 1.2 (n = 3)	Mid to high concentration range (200 µg/L)
Truenesstestsample and mass balance measurements	Quality control standard (Merck XXI): Fe: 98.7 ± 2.4 (n = 27) Mg: 99.4 ± 1.8 (n = 27)	High concentration range (100 µg/L)
Truenesstestsample and mass balance measurements	Quality control standard (Merck XXI): Fe: 100 ± 1.5 (n = 12) Mg: 101 ± 1.7 (n = 12)	High concentration range (500 µg/L)
Truenesstestsample measurements	Quality control standard (Merck XXI): Fe: 96.4 ± 0.6 (n = 5) Mg: 97.8 ± 0.6 (n = 5)	High concentration range (1000 µg/L)
Truenesstestsample and mass balance measurements	Recalibration standard Fe: 98.9 ± 2.4 (n = 19) Mg: 104 ± 4.2 (n = 19)	Low to Mid concentration range (50 µg/L)
Truenesstest sample measurements	Recalibration standard Fe: 98.2 ± 1.7 (n = 8) Mg: 97.3 ± 1.9 (n = 8)	Mid to high concentration range (100 µg/L)
Truenesstestsample and mass balance measurements	Recalibration standard Fe: 100 ± 2.2 (n = 17) Mg: 98.2 ± 2.3 (n = 17)	High concentration range (250 µg/L)
Trueness test samples	Fortification of samples: Fe: 92.9 - 112 % Mg: 99.3 – 114 %
Truenessmass balancesample measurements	Fortification of samples: Fe: 98.9 – 101 %

Solution pH values

The target pH in all media before addition of test substance was in the nominal range.

During the study, the pH of GST, ALF and PBS media remained stable in the method blank vessels and the test vessels. Therefore, a possible effect of the test substance can be excluded. In GMB medium, the pH in all vessels (including method blanks) increased during the time of the test from 7.40 to 8.77, 8.33 and 8.80 (test vessels) and 7.40 to 8.79 and 8.81 (method blank vessels). Therefore, an effect of the test substance can be excluded. In fact, the pH of the GMB media does not seem to be stable under the conditions of the test. In ASW medium, the pH in all vessels (including method blanks) decreased during the time of the test from 6.50 to 5.91, 5.92 and 5.93 (test vessels) and 6.50 to 5.96 and 5.99 (method blank vessels). Therefore, an effect of the test substance can be excluded.

Temperature control

The test was performed in an incubated laboratory shaker (Shaking incubation cabinet, Minitron, INFORS AG, Bottmingen, Switzerland) at 100 rpm. The temperature was adjusted to 37.5 °C in a thermostatically controlled shaking cabinet to reach a temperature of 37 °C ± 2 °C in the media. The temperature remained stable during the test in all media.

Fortification

For fortified test samples, recoveries were in the range of 92.9 – 112 % for Fe and the recoveries of Mg were in the range of 99.3 – 114 % in test samples.

Fortification of test samples with Iron

sample	measured concentration of Fe [µg/L]	calculated level after addition [µg/L]	recovery [µg/L] / [%]
GST vessel 1 sample a 2h	78.6	35.7	35.7 / 99.9
GST vessel 1 sample a 24h	467	113	119 / 105
GMB vessel 2 sample a 2h	<LOD	15.1#	15.8 / 105
GMB vessel 1 sample a 24h	<LOD	14.6	16.3 / 112
ASW vessel 1 sample a 2h	<LOD	8.00	8.10 / 101
ASW vessel 1 sample a 24h	4.04	8.81	8.93 / 101
ALF vessel 2 sample b 2h	63.4	92.7	93.2 / 101
ALF vessel 2 sample b 24h	217	123	122 / 99.1
PBS vessel 1 sample a 2h	<LOD	14.2	14.2 / 100
PBS vessel 2 sample a 24h	<LOD	4.61	4.28 / 92.9

^#Solutions with concentrations below the LOD/LOQ were also fortified. However, a recovery of ± 15 % may not be realistic as concentrations in the original (unfortified) sample below the LOD may be lower than the noise ratio and concentrations below the LOQ are between the noise ratio and a real (quantifiable) signal. For solutions with concentrations below the LOD/LOQ, a recovery of ± 25 % - 30 % is more realistic.

Fortification of test samples with magnesium

sample	measured concentration of Mg [µg/L]	calculated level after addition [µg/L]	recovery [µg/L] / [%]
GST Vessel 1 sample a 2h	3.19	20.6	20.9 / 101
GST Vessel 1 sample a 24h	8.22	21.6	22.1 / 102
GMB vessel 2 sample a 2h	<LOD	14.8	15.6 / 105
GMB vessel 1 sample a 24h	<LOD	14.6	15.8 / 108
ASW vessel 1 sample a 2h	0.80	8.16	8.99 / 110
ASW vessel 1 sample a 24h	2.87	8.57	9.38 / 109
ALF vessel 2 sample b 2h	3.36	80.7	81.0 / 100
ALF vessel 2 sample b 24h	5.39	81.1	80.5 / 99.3
PBS vessel 1 sample a 2h	<LOD	15.0	15.7 / 105
PBS vessel 2 sample a 24h	<LOD	6.00	6.86 / 114

For fortified test samples, recoveries were in the range of 98.9 – 101 % for Fe in mass balance samples.

 

Fortification of mass balance samples with iron.

sample	measured concentration of Fe [µg/L]	calculated level after addition [µg/L]	recovery [µg/L] / [%]
ALF vessel 1 (dilution factor 100)	56.9	64.1	65.0 / 101
ASW vessel 1 (dilution factor 100)	126	175	175 / 100
GMB vessel 1 (dilution factor 100)	249	349	346 / 99.0
GST vessel 1 (dilution factor 100)	105	163	161 / 98.9
PBS vessel 1 (dilution factor 100)	134	180	179 / 99.3

Conclusions:

On the basis of OECD Series on Testing and Assessment No. 29 as well as according to a bioaccessibility test protocol, which has been developed on the basis of relevant published methods, the dissolution of the pigment IPC-2018-008 (magnesium ferrite) in the artificial physiological media (GST, GMB, ALF, ASW and PBS) with a single loading of 100 mg/L, agitation (100 rpm) at 37 °C ± 2 °C and sampling after 2 and 24 h, was determined. The measurement of dissolved iron and magnesium concentrations after filtration were performed by ICP-OES. The study was performed in triplicate with two additional method blanks per medium.

Concentration of dissolved iron in artificial physiological media.
Total Fe ± SD in sample vessels
- GST 2h:75.5± 8.67 µg/L
- GST 24h: 474 ± 17.1 µg/L
- GMB 2h: 0.265 ± 0.213
- GMB 24h: All samples below LOD
- ALF 2h: 66.9 ± 4.50 µg/L
- ALF 24h: 107.246 ± 2.25 µg/L
- ASW 2h: All samples below LOD
- ASW 24h: 0.802 ± 1.12
- PBS 2h: All samples below LOD
- PBS 24h: All samples below LOD

Concentration of dissolved zinc in artificial physiological media.
Total Zn ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with mean method blank subtraction (mean of 2 h and/or 24 h)
- GST 2h: 3.30 ± 0.62 µg/L
- GST 24h: 2.17± 0.51 µg/L
- GMB 2h: All samples below LOD
- GMB 24h: All samples below LOD/LOQ
- ALF 2h: 2.18 ± 1.51 µg/L
- ALF 24h: 1.07 ± 0.41 µg/L
- ASW 2h: 0.55 ± 0.23 µg/L
- ASW 24h: 0.44 ± 0.32 µg/L
- PBS 2h: All samples below LOD
- PBS 24h: All samples below LOD/LOQ


Concentration of dissolved magnesium in artificial physiological media.
Total Mg ± SD in sample vessels with mean method blank subtraction (mean of 2 h and/or 24 h)
GST 2h: 174 ± 3.09 μg/L
-GST 24h: 242 ± 3.96 μg/L
-GMB 2h: 153 ± 48.8 μg/L
-GMB 24h: 138 ± 18.4 μg/L
-ALF 2h: 297 ± 105 μg/L
-ALF 24h: 23.2 μg/L
-ASW 2h: 78.0 ± 5.75 μg/L
-ASW 24h: 167 ± 6.31 μg/L
-PBS 2h: 110 ± 2.94 μg/L
-PBS 24h: 161 ± 2.05 μg/L

Description of key information

In conclusion, since the dissolved Fe and Mg concentrations from this pigment under simulated physiological conditions were below 468 µg/L and 4.46 µg/L (GST, ALF), respectively even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.5% after 24 hours, this pigment may reasonably be considered biologically inert.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

The chemical and physiological properties of the pigment Magnesium ferrite are characterised by inertness because of the specific synthetic process (calcination at high temperatures, approximately 1000°C), rendering the substance to be of a unique, stable crystalline structure in which all atoms are tightly bound and not prone to dissolution in environmental and physiological media. This manufacturing process leads to a very low bioaccessibility of the elements contained in the pigment. This has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation), as follows:

 

1.) Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions,

2.) phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum,

3.) artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating,

4.) artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and

5.) artificial gastric fluid (GST, pH 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.

Solubility of Fe from the pigment Magnesium ferrite in physiological media was in a range of below LOD(ASW, PBS) and 74.6 µg/L (GST) after 2 hours. After 24 hours a dissolution range from below LOD (PBS, GMB) - 468 µg/L (GST) was determined.

Solubility of Zn from the pigment Magnesium ferrite in physiological media was in a range of below LOD(GMB, PBS) and 2.80 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from below LOD (GMB, PBS) - 2.18 µg/L (ALF) was measured.

Solubility of Mg from the pigment Magnesium ferrite in physiological media was in a range of 78 µg/L (ASW) and 297 (ALF) µg/L after 2 hours. After 24 hours a dissolution range from below 110 µg/L (PBS) and 242 µg/L (GST) was measured.

In conclusion, since the dissolved Fe, Mg and Zn concentrations from this pigment under simulated physiological conditions were below 468 µg/L, 242 µg/L

and 2.80 µg/L (GST, ALF), respectively even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.5% after 24 hours, this pigment may reasonably be considered biologically inert.